Cell Immunotherapy for Cancer
The following methods are known as cell immunotherapy methods for the treatment of cancer (Choi D et al., Clin. Cancer Res., 4, 2709-2716, 1998, Hsu F J et al., Nat Med., 2, 52-58, 1996, Takamizawa M et al., J Clin. Invest 95, 296-303, 1995, Reichardt V L et al., Blood, 93, 2411-2419, 1999, wang J et al., J. Immunol., 161, 5516-5524, 1998, Morse M A et al., Clin. Cancer Res., 5, 1331-1338, 1999). Cells obtained by selectively collecting dendritic cells (DC)/macrophages by apheresis are further cultured in a culture liquid to which has been added interleukin 4, GMCSF and TNF-α to induce differentiation (Steinman R M et al., Adv. Exp. Med.1997). Subsequently, the cells are detached and recovered from the culture vessel by any method of (i) enzyme treatment, (ii) scraping with a scraper, or (iii) blowing the culture liquid with a pipette (Hodes R J and Singer A., Eur. J. Immunol., 7, 892-897, 1977). Mature DC/macrophages obtained in this manner are then co-cultured in medium containing a target antigen to produce cancer-specific antigen-presenting cells having a complex of target antigen epitope and MHC class II molecules. The antigen-presenting cells adhered to the bottom of the culture vessel are detached and recovered by any method of (i) enzyme treatment, (ii) scraping with a scraper, or (iii) blowing on the culture liquid with a pipette, followed by administering to the same person in the form of cancer-specific, antigen-presenting cells.
In conventional immune cell therapy, cultured cells were detached and recovered from a culture vessel by any method of (i) enzyme treatment, (ii) scraping with a scraper, or (iii) blowing on the culture liquid with a pipette (Hodes RJ and Singer A., Eur. J. Immunol., 7, 892-897, 1977).
Detachment and Recovery Methods Using Enzymes
A method using trypsin is most commonly used for detaching and recovering adhered cells (Hodes R J and Singer A., Eur J. Immunol., 7, 892-897, 1977). In this method, after washing the culture vessel to which the cells are adhered with PBS not containing Ca++ or Mg++, PBS containing 0.05% trypsin and 0.024% EDTA is added to a depth of 0.1 to 1 mm from the bottom of the flask and allowed to stand undisturbed at room temperature. 5 to 10 minutes later, culture liquid containing 0.1 to 1% albumin chilled to 4° C. is added followed by stirring gently to gather the suspended cells. The gathered cells are then washed with culture liquid containing albumin for use in subsequent experiments.
However, detaching methods using enzymes have a problem in that, since functional protein molecules on the cell membrane of the adhesive immune cells are digested and decomposed, even if they are detached and recovered, they are unable to adequately demonstrate cell functions. Although methods using a Teflon film and so forth for the culture vessel have been previously attempted (Van der Meer J W M, Bulterman D., Van Zwet T L, Elzenga-Claasen I, and Van Furth R, J. Exp. Med., 147, 271-276, 1978; Van der Meer J W M, Van de Gevel J S, Elzenga-Claasen I, and Van Furth R, J, Cell Immunol., 42, 208, 1979), since adhesion to the culturing surface is obstructed, it was difficult to acquire the function by culturing. Consequently, there is a need to develop a culture base having the offsetting properties of maintaining adhesion during culturing, but losing affinity for cells during detaching and recovery.
An object of the present invention is to provide adoptive immune cells capable of adequately demonstrating cell functions by using a culture base that solves the aforementioned problems.